Project Summary Discovered four years ago, Orsay is the first and also the only virus known to naturally infect the model nematode species Caenorhabditis elegans (C. elegans). Orsay has a bi-segmented, plus-sense RNA genome that expresses a total of four polypeptides, including an RNA-dependent RNA polymerase (RdRP), a capsid protein (CP), a nonstructural protein ?, and a CP-? fusion protein produced by ribosomal frameshift. Recently we determined the crystal structure of an Orsay virus-like particle, which shows a CP structural organization that is related to betanodaviruses but distinct from those insect-infecting alphanodaviruses. Considering the simplicity of Orsay and the ease of laboratory manipulation of C. elegans as a popular model organism, Orsay-C. elegans enables detailed mechanistic studies on virus replication, virus-host interaction and host innate antiviral responses in an intact organism using a natural pathogen. Orsay infection occurs primarily in intestine cells causing abnormal intestinal morphology. Our preliminary studies of the Orsay ?/CP-? proteins have generated exciting results indicating potential roles in cell entry and also non-lytic viral release by restructuring the host cytoskeleton network. We showed that overexpressed ? in transgenic worms was mainly localized to/near the apical membrane of the animal intestine. Orsay-infected worms displayed disorganized actin network that is associated with the apical microvilli in intestinal cells. Similarly disorganized actin network was observed in transgenic worms following ? protein expression alone. Recombinant ? protein folds into a fibrous molecule and co-purifies with several host cytoskeleton proteins. To elucidate how Orsay interacts with the C. elegans host to ensure successful replication, the two laboratories of Yizhi Jane Tao and Weiwei Zhong, with expertise in structural virology and C. elegans genetics, respectively, will collaborate to determine the structure and the biological activities of the Orsay ?/CP-? proteins and to discover the nematode antiviral gene network using high-throughput, genome-wide screens based on a novel phenotype-based assay. Findings from our study will likely shed new light on the infection mechanisms used by other gastrointestinal viruses such as rotavirus, astrovirus, norovirus, etc. Specific Aim 1: Characterize the structure of the Orsay ? and CP-? proteins. We plan to determine the structure of both proteins using the combination of X-ray crystallography and cryo-EM. Specific Aim 2: Characterize Orsay-host interaction mediated by ?/CP-?. We plan to identify host proteins that physically interact with ? using co-IP, and to characterize the molecular nature and biological implications of such interaction both in vitro and in vivo. Specific Aim 3: Discover host antiviral gene networks. We will carry out a genome-wide screen for antiviral genes, perform phenotypic characterization of antiviral genes, and map antiviral pathways using both computational and experimental approaches.